This study investigated the use of "naturalistic stressors" such as physical restraint and animal pheromones on the etiology of Posttraumatic Stress Disorder in a murine model. Pilot data suggest that stress effects may lead to an increase in the amount of time needed for cutaneous wounds to heal. Pilot data to support the creation of this model are presented suggesting that a delayed stress response may inhibit healing rates. In the present study an animal model of PTSD was used to investigate the effect of stress on the immune system. Yehuda and Antelman's (1993) nonhuman animal model of Posttraumatic Stress Disorder was tested with respect to the animals' immune response to cutaneous wounding. Additionally, effects of stress on exploratory behavior and activity were examined. The findings support the hypothesis that restraint and pheremonal stress and housing arrangements influence the ability of mice to heal a 1.5 mm punch biopsy, and exploratory behavior. The findings also support a profile for the Post-Traumatic Mouse. / Ph. D.

A practical consideration in the development of cellular therapy technology for the inner ear is the development of an in vitro model for assessing the optimal conditions for successful application of cells. The first part of this thesis describes the adaptation of the cochleovestibular structure harvested from P1 mouse pups for analysis of factors critical for the optimal implantation of stem cells in the inner ear. Results of these studies establish that the c17.2 neural stem cell line can be introduced into the cochleovestibular structure in vitro. Using this model, c17.2 cells demonstrated survival predominantly within the vestibule and basal spiral ganglion regions. Furthermore, the addition of the ototoxin, cisplatin and the neurotrophin, Brain Derived Neurotrophic Growth Factor (BDNF) enhanced the survival and migration/dispersion of c17.2 cells within the cochleovestibular explant. The second part of this thesis examines the hypothesis that olfactory neurosphere (ONS) and progenitor cells harvested from the olfactory epithelium represent a viable source of graft material for potential therapeutic applications in the inner ear. Olfactory epithelium represents a unique source of pluripotent cells that may serve as either homografts or autografts. The feasibility of ONSs to survive and integrate into a mammalian cochlea in vivo was assessed. The ONSs were isolated as a crude fraction from the olfactory epithelium of P1 to P3 day old swiss webster mouse pups, ubiquitously expressing the Green Fluorescent Protein (GFP) marker. The ONSs were microinjected into the cochleae of adult CD1 male mice. Four weeks following their implantation, ONS cells expressing the GFP marker and stained by Nestin were identified in all areas of the cochlea and vestibule, including the spiral ganglion. Robust survival and growth of the implanted ONS and ONS derived cells in the cochlea also included the development of ???tumor-like??? clusters, a phenomenon not observed in control animals implanted with c17.2 neural stem cells. Collectively, the results of this thesis illustrate the potential of olfactory neurosphere and progenitor cells to survive in the inner ear and expose a potential harmful effect of their transplantation.

Alzheimer’s disease (AD) is the most commonly diagnosed form of dementia in the aged, and is characterised by a progressive decline in memory, language and other cognitive functions, together with deterioration in behavioural, emotional and social skills. The earliest clinical symptoms include episodic memory loss and dysnomia. This is followed by other signs of cortical impairment including apraxia, agnosia, and visuospatial impairment. In advanced stages, victims become mute, cannot walk and are incontinent; they therefore become totally dependent on carers. AD is the third leading cause of death in the aging population after heart disease and cancer. The incidence of AD doubles every 5 years in subjects between the ages of 65 and 85 years, affecting one in three by the age of 80. AD is characterised by the existence of intracellular and extracellular amyloid deposits in the brain. Extracellular amyloid deposits consist of plaques, whereas the deposits within and around blood vessels are referred to as cerebral amyloid angiopathy (CAA). Neurofibrillary tangles (NFT) are characteristically found in AD; however, they are also found in some other neurodegenerative disorders such as tuberose sclerosis, amyotrophic lateral sclerosis, parkinson-dementia complex and dementia pugilistica.

Peptide YY (PYY) is a gut-derived hormone that is renowned for its effects on satiety. Reduced satiety in obese people has been attributed to low fasting and postprandial PYY levels. However, it has not been determined whether low PYY levels are the cause or the outcome of obesity. Moreover, the long-term role of PYY in regulating energy balance is unclear. Results presented in this thesis, using PYY-deficient mice (PYY-/-) and PYY transgenic mice (PYYtg) highlight that PYY indeed has an important role in regulating energy balance and glucose homeostasis in vivo. PYY knockout mice became obese with ageing or high-fat feeding linked to a hyperinsulinemic phenotype associated with hypersecretion of insulin from isolated pancreatic islets. These findings suggested that PYY deficiency may be a predisposing factor for the development of obesity and type 2 diabetes. On the other hand, PYYtg mice exhibited decreased adiposity and increased metabolism under high-fat feeding. Furthermore, PYYtg/ob mice had improved glucose tolerance and decreased adiposity. These latter studies suggested that high circulating PYY levels may protect against the development of obesity and type 2 diabetes. Interestingly, both animal models support PYY as an important regulator of the somatotropic axis. These preliminary findings prompted investigations in understanding whether low PYY levels may be a predisposing factor for the development of obesity and type 2 diabetes in human subjects. In a population of healthy human subjects that had a predisposition to the development of type 2 diabetes and obesity, fasting PYY levels were lower than in normal subjects. Moreover, low fasting PYY levels strongly correlated with decreased insulin sensitivity and high levels of fasting insulin. Collectively, these findings suggest that low circulating levels of PYY could contribute to increased adiposity, insulin resistance and type 2 diabetes. Therefore determination of PYY levels may be a method of detecting whether people are predisposed to becoming obese and insulin resistant. This work also suggests that treatments that enhance circulating PYY levels may be protective in the development of obesity and type 2 diabetes.

published_or_final_version / Anaesthesiology / Master / Master of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Several trace metals have been shown to modify cell injury as indicated by reduction of observable pathological tissue changes after metal ion supplementation. An example of this is zinc ion induced protection against some of the liver injury caused by a single injection of carbon tetrachloride (CCl₄) to male Sprague-Dawley rats. Carbon tetrachloride liver injury is associated with membrane labilization as indicated by lysosomal and endoplasmic reticulum anomalies. Depressed hepatic levels of NADPH are observed during CCl₄ poisoning. Lipid metabolism is also impaired due to CCl₄ administration to animals. These biochemical manifestations of CCl₄ hepatotoxicity were studied in the presence of zinc ions in order to understand the mechanisms of the zinc protective effect against CCl₄ injury. The effect of zinc ions on the stability of rat liver lysomes was studied. Zinc was added by several methods: (1) feeding the animals a high zinc diet, (2) infusion of zinc into the liver in situ through the portal vein, or (3) by adding zinc to the lysosomal fraction either before or after isolation of this fraction from rat liver homogenates. By all techniques, addition of zinc reduced the release of β-glucuronidase from liver lysosomes, indicating increased stability of the suborganelles. The zinc induced protection against CCl₄ liver damage was evident in observations made using both light microscopy and electron microscopy. The increased release of lysosomal β-glucuronidase observed in the CCl₄ treated rats was significantly reduced by zinc administration. Also, decreases in microsomal protein synthesis and seromucoid levels due to the CCl₄ treatment were significantly ameliorated by zinc. Thus, disruption of lysosomal and endoplasmic reticulum membranes, one of the earliest signs of CCl₄ hepatotoxicity, appeared to be inhibited by pretreating the animals with zinc chloride. Depressed hepatic levels of NADPH are observed in rats administered CCl₄. Zinc chloride pretreatment of these rats significantly increased the NADPH levels in the liver. Since zinc ions bind NADPH, then the protective effect of zinc against CCl₄ toxicity may be due to stabilization of the pyridine nucleotide by zinc and the subsequent prevention of CCl₄ induced alterations of biochemical reactions dependent upon NADPH. Zinc chloride pretreatment of CCl₄ treated rats significantly reduced the CCl₄ induced hepatic triacylglycerol accumulation. Concomitant with this event is the appearance of elevated levels of newly synthesized triacylglycerols in the serum of the CCl₄ treated rats given zinc above that of the CCl₄ treated rats. Hepatic triacylglycerol synthesis is unchanged by CCl₄ or zinc treatment. Hepatic phospholipid levels which are depressed by the CCl₄ hepatotoxin are not affected by zinc treatment. However, the synthesis of phospholipids in the livers of rats treated with CCl₄ plus zinc is significantly increased. The lipid changes induced by CCl₄ and zinc dosing of rats are indicative of alterations in liver membranes thus affecting hepatic liver transport mechanisms. On the basis of the data presented, the effects of zinc ions on CCl₄ hepatotoxicity are discussed and applied to understanding the regulating role of metal ions in tissue injury. The protective effects of zinc ions against CCl₄ hepatotoxicity suggest a relationship between the zinc nutritional status of animals exposed to environmental contaminants, and the expression of the ensuing tissue damage.

Systemic Lupus Erythematosus (SLE) is a multi-organ autoimmune disorder that may result in death due to cardiac dysfunction. This dysfunction often occurs due to an endocarditis, known as Libman-Sacks, which presents on heart valves. The condition is hard to clinically diagnose and is often observed postmortem. Heart damage has been observed in the NP-SLE Lewis rat model positive for SLE. However, research has not been done in this model on the correlation between SLE and Libman-Sacks endocarditis. Numbers of occurrence have ranged from 3-50% in SLE patients. The presence of Libman-Sacks endocarditis should likewise occur in 3-50% of NP-SLE Lewis rats. There will be seven NP-SLE Lewis rats, five negative serum control rats, and five saline injected control rats. By performing this controlled study in rats, the correlation between SLE and Libman-Sacks will be better understood. / Department of Physiology and Health Science

A practical consideration in the development of cellular therapy technology for the inner ear is the development of an in vitro model for assessing the optimal conditions for successful application of cells. The first part of this thesis describes the adaptation of the cochleovestibular structure harvested from P1 mouse pups for analysis of factors critical for the optimal implantation of stem cells in the inner ear. Results of these studies establish that the c17.2 neural stem cell line can be introduced into the cochleovestibular structure in vitro. Using this model, c17.2 cells demonstrated survival predominantly within the vestibule and basal spiral ganglion regions. Furthermore, the addition of the ototoxin, cisplatin and the neurotrophin, Brain Derived Neurotrophic Growth Factor (BDNF) enhanced the survival and migration/dispersion of c17.2 cells within the cochleovestibular explant. The second part of this thesis examines the hypothesis that olfactory neurosphere (ONS) and progenitor cells harvested from the olfactory epithelium represent a viable source of graft material for potential therapeutic applications in the inner ear. Olfactory epithelium represents a unique source of pluripotent cells that may serve as either homografts or autografts. The feasibility of ONSs to survive and integrate into a mammalian cochlea in vivo was assessed. The ONSs were isolated as a crude fraction from the olfactory epithelium of P1 to P3 day old swiss webster mouse pups, ubiquitously expressing the Green Fluorescent Protein (GFP) marker. The ONSs were microinjected into the cochleae of adult CD1 male mice. Four weeks following their implantation, ONS cells expressing the GFP marker and stained by Nestin were identified in all areas of the cochlea and vestibule, including the spiral ganglion. Robust survival and growth of the implanted ONS and ONS derived cells in the cochlea also included the development of ???tumor-like??? clusters, a phenomenon not observed in control animals implanted with c17.2 neural stem cells. Collectively, the results of this thesis illustrate the potential of olfactory neurosphere and progenitor cells to survive in the inner ear and expose a potential harmful effect of their transplantation.

Peptide YY (PYY) is a gut-derived hormone that is renowned for its effects on satiety. Reduced satiety in obese people has been attributed to low fasting and postprandial PYY levels. However, it has not been determined whether low PYY levels are the cause or the outcome of obesity. Moreover, the long-term role of PYY in regulating energy balance is unclear. Results presented in this thesis, using PYY-deficient mice (PYY-/-) and PYY transgenic mice (PYYtg) highlight that PYY indeed has an important role in regulating energy balance and glucose homeostasis in vivo. PYY knockout mice became obese with ageing or high-fat feeding linked to a hyperinsulinemic phenotype associated with hypersecretion of insulin from isolated pancreatic islets. These findings suggested that PYY deficiency may be a predisposing factor for the development of obesity and type 2 diabetes. On the other hand, PYYtg mice exhibited decreased adiposity and increased metabolism under high-fat feeding. Furthermore, PYYtg/ob mice had improved glucose tolerance and decreased adiposity. These latter studies suggested that high circulating PYY levels may protect against the development of obesity and type 2 diabetes. Interestingly, both animal models support PYY as an important regulator of the somatotropic axis. These preliminary findings prompted investigations in understanding whether low PYY levels may be a predisposing factor for the development of obesity and type 2 diabetes in human subjects. In a population of healthy human subjects that had a predisposition to the development of type 2 diabetes and obesity, fasting PYY levels were lower than in normal subjects. Moreover, low fasting PYY levels strongly correlated with decreased insulin sensitivity and high levels of fasting insulin. Collectively, these findings suggest that low circulating levels of PYY could contribute to increased adiposity, insulin resistance and type 2 diabetes. Therefore determination of PYY levels may be a method of detecting whether people are predisposed to becoming obese and insulin resistant. This work also suggests that treatments that enhance circulating PYY levels may be protective in the development of obesity and type 2 diabetes.